4 molecular marker assay

A generalized molecular marker refers to a heritable and detectable DNA sequence or protein. Protein markers include seed storage proteins and isozymes (different molecular forms of enzymes encoded by more than one locus) and allozymes (different molecular forms of enzymes encoded by different alleles of the same locus) . The narrow concept of molecular markers only refers to DNA markers, and this definition is now widely adopted. DNA polymorphism is a technique for detecting polymorphisms due to deletions, insertions, translocations, inversions, rearrangements, or lengths and sequences of repeated sequences in DNA molecules. Therefore, DNA molecular marker technology is also called molecular diagnostic technology. Molecular markers can reflect the DNA fragments of a particular feature in the individual's or population's genome. It directly reflects the differences between genomic DNA. Compared with the above three identification methods, molecular markers have many obvious advantages. They are: (1) directly expressed in the form of DNA and can be detected in all tissues and developmental stages of organisms, regardless of seasons and environmental restrictions. , There are no problems such as expression or not; (2) The number is extremely high, which spreads over the whole genome, and the detectable seats are almost infinite; (3) There are many polymorphisms in nature, there are many allelic variations in nature, and no artificial creation is required; (4) Neutrality does not affect the expression of the target traits; (5) Many markers are characterized by codominance and can distinguish between homozygotes and heterozygotes, providing complete genetic information. These advantages of molecular markers have made it widely used in the construction of plant molecular maps, plant genetic diversity analysis and germplasm identification, important agronomic trait gene mapping and map cloning, identification of transgenic plants, and sub-marker-assisted breeding selection and other life sciences. Theory and applied research.

In the long-term evolution of organisms, there are many heritable variations caused by DNA base sequence variation. These DNA base sequence variations mainly include DNA rearrangements, base substitutions, deletions, insertions, inversions, translocations, and sequence repeats. Molecular marker technology is based on this extremely rich DNA base sequence variation in the species.

4.1 RFLP (Restration fragment length polymorphism, restriction fragment length polymorphism marker)

This technique was founded by Grodzicker et al. in 1974 and again by Botstein in 1980. It was first applied by SOLLER and Botstein (1983) for the identification of species and the determination of the purity of lines, which was the first DNA molecular marker applied to genetic research. technology.

RFLP is a genetic marker developed on the basis of molecular cloning techniques. The genomic DNA of a specific biological type, after being completely digested by a restriction endonuclease, will produce homologous allele fragments with different molecular weights, or restriction alleles. The basic principle of RFLP labeling technology is to separate and detect these fragments by electrophoresis. All mutations that can cause mutations in the enzymatic sites, such as point mutations (new generation and deletion sites) and a reorganization of DNA (such as insertions and deletions that change the length of the cleavage site), can be This leads to changes in the restriction alleles, resulting in RFLP polymorphisms. The technology consists of the following basic steps: DNA extraction; digestion with DNA restriction enzymes; separation of restriction fragments by gel electrophoresis; transfer of these fragments in their original sequence and position to a disposable filter; use radioisotopes or non- The radioactive material labeled DNA is used as a probe to hybridize with the DNA on the membrane (called Southern hybridization); autoradiography or enzymatic detection shows that the different materials of the probe's restriction fragment polymorphism can be generated and obtained. Reflects individual or population-specific RFLP patterns. The probe used in the RFLP analysis is usually a single-copy or low-copy genomic fragment or cDNA fragment randomly cloned and having certain homology with the test substance.

The advantages of RFLP fingerprinting are mainly reflected in: First, the reliability is high. Because it is produced by restriction endonuclease cleavage of specific sites; second, it is derived from natural variations. There is no need for any mutagen treatment based on the abundant base variation in DNA; third, diversity. All the differences are reflected at the DNA level by the enzymatic reaction, and the RFLP markers are distributed throughout the entire genome, so there is almost no limit in the number; fourth, codominance. RFLP markers distinguish between heterozygotes and homozygotes; fifth, no phenotypic effects. Not limited by developmental stage and organ specificity.

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